The present invention relates to a new and improved method of, and apparatus for, producing a relief-like contrast at a microscope image or micrograph of a transparent phase object.
During the observation of a transparent, non-contrasting phase object, especially living tissues or cells, it is already known to the art for generating a relief-like contrast at the microscope image of such object to illuminate the object obliquely with regard to the object plane at one side from the underside of the object slide or support. In the context of this disclosure there is to be understood under the term "phase object" a microscope object wherein the optical distance is locally inhomogeneous.
However, with this relatively simple technique there can only be realized a relatively low resolution. The reason for the poor resolution is predicated upon the diffraction effects which cannot be avoided when employing conventional illumination techniques and which practically render impossible the observation of smaller structures.
The development of the interference microscope constituted a measure which attempted to overcome this situation. As is known the most commonly used interference microscopes, for instance those working according to the principles of Jamin-Lebedeff and those according to Nomarski, work with linear polarized light which is split at a so-called Wollaston-prism into two light beams which are polarized at right angles to one another and spatially parallelly offset. After both of the beams of light rays have passed through the phase object and the objective they are again consolidated at a second Wollaston-prism and with the aid of an analyzer brought to interference. The thus prevailing intermediate image is then observed in conventional manner by means of an ocular. The interference image possesses a characteristic plastic effect which is similar to the one-sided oblique illumination of the bright ground or field.
Unfortunately it has however been found that although the principle of operation of an interference microscope is indeed relatively simple, still the practical construction of such microscope is associated with considerable difficulties. The fact that it is particularly necessary to work with polarized light especially contributes to these difficulties. As is well known it is almost virtually impossible to mount the optical lenses at the microscope free of stress, with the result that undesired double refraction effects arise which again must be avoided in a quite cumbersome manner. Moreover, it is extremely difficult to fabricate a Wollaston-prism from which emanate with the same intensity the ordinary and extraordinary light beams. Hence, technical difficulties arise during the construction of an interference microscope which, although capable of being overcome with the technology available at the present time, still nonetheless considerably increase the price of such microscope.
The microscopes used for scientific purposes generally possess an illumination device which transmits the light from a light source via a condensor onto the object which is to be observed. During the course of the development of microscopes it has been found that the condensor is responsible for a great many image errors. Therefore, attempts have of course been made to continually improve upon the condensors and it has actually been possible to overcome numerous disturbing effects. Yet, no condensor which has been developed up to the present time can avoid diffraction effects of object structures from becoming visible at the image plane and which are not focused at the image plane. These diffraction patterns are superimposed upon the image of the phase object and render interpretation of the image more difficult.